Project Summary: Clinical and basic scientific evidence indicates that alterations in the brain's inhibitory mechanisms are critically important to both the causes and treatments for epilepsy. Despite recent major advances in understanding the biology of synaptic transmission, very little is known about the adaptive processes that balance inhibitory control mechanisms with excitatory drive. GABA synthesis in inhibitory neurons is regulated by the supply of its precursor glutamate through direct uptake via Excitatory Amino Acid Transporter 3 (EAAT3], and uptake of glutamine, which can be deamidated in neurons to produce glutamate, via Sodium-coupled Neutral Amino Acid Transporter 1 (SNATl). Substrate supply through these transporters bidirectionally regulates the synaptic vesicle content of GABA and the strength of inhibitory synaptic transmission. The central hypothesis of this proposal is that a dynamic regulation of synaptic inhibition is mediated by the supply of substrates for GABA metabolism. We further hypothesize that the EAAT3-mediated regulation of inhibition selectively enhances inhibitory circuits that counterbalance hyperexcitability for the prevention of seizures in the hippocampus. This proposal has three specific aims: In the first aim, we will investigate developmental changes in the contribution of SNATl and EAAT3 to the regulation of Inhibition at specific subsets of synapses. In the second aim, we will investigate the other key factors such as transporter properties and substrate availability that determine the relative contributions of glutamine and glutamate to the regulation of synaptic inhibition. In the third specific aim, the effect of targeted EAAT3 over-expression in epileptic hippocampus will be investigated to evaluate the compensatory role of pre-synaptic glutamate uptake in an animal model of human mesial temporal lobe epilepsy. We expect these studies to demonstrate that GABA metabolism and synaptic inhibition are dynamically regulated by substrate supply through transporters and that this regulation plays an important role in maintaining normal function in the hippocampus. Public Health Relevance: Ultimately, these studies will identify the key regulatory molecules in GABA's metabolic pathways for further evaluation as new susceptibility factors for epilepsy as well as targets for novel therapeutic interventions for the treatment of intractable epilepsy.